1. Field of the Invention
xe2x80x9cThis 371 application claims the benefit of PCT/FR94/00422, filed Nov. 10, 1994, which claims the benefit of French application FR93/05125, filed Apr. 30, 1993.xe2x80x9d
The present invention relates to new recombinant viruses, to their preparation and to their use in gene therapy, for the transfer and expression in vivo of desired genes. More specifically, it relates to new recombinant viruses comprising an inserted gene whose expression in vivo makes it possible to regulate the plasma levels of apolipoproteins. The present invention also relates to pharmaceutical compositions comprising the said recombinant viruses. More particularly, the present invention relates to defective recombinant viruses and to their use for the prevention or treatment of pathologies linked to dyslipoproteinemias which are known for their serious consequences at the cardiovascular and neurological level.
2. Description of Related Art
Dyslipoproteinemias are disorders of the metabolism of the lipoproteins responsible for the transport, in the blood and peripheral fluids, of lipids such as cholesterol and triglycerides. They result in major pathologies, linked respectively to hypercholesterolemia or hypertriglyceridemia, such as especially atherosclerosis. Atherosclerosis is a polygenic complex disease which is defined from the histological point of view by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives in the wall of the large arteries (aorta, coronary arteries, carotid). These plaques, which are calcified to a greater or lesser extent according to the progression of their process, can be associated with lesions and are linked to the accumulation, in the arteries, of fatty deposits consisting essentially of cholesterol esters. These plaques are accompanied by a thickening of the arterial wall, with hypertrophy of the smooth muscle, the appearance of spumous cells and the accumulation of fibrous tissue. The atheromatous plaque is very clearly in relief on the wall, which confers on it a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism which occur in the patients most affected. Hypercholosterolemias can therefore result in very serious cardiovascular pathologies such as infarct, sudden death, cardiac decompensation, cerebral vascular accidents and the like.
It is therefore particular important to be able to have available treatments which make it possible to reduce, in certain pathological situations, the plasma cholesterol levels or even to stimulate the efflux of cholesterol (reverse transport of the cholesterol) in the peripheral tissues in order to discharge the cells having accumulated cholesterol within the context of the formation of an atheroma plaque. The cholesterol is carried in the blood by various lipoproteins including the low-density lipoproteins (LDL) and the high-density lipoproteins (HDL). The LDLs are synthesized in the liver and make it possible to supply the peripheral tissues with cholesterol. In contrast, the HDLs capture cholesterol in the peripheral tissues and transport it to the liver where it is stored and/or degraded.
At present, dyslipemias and in particular hypercholesterolemias are treated essentially by means of compounds which act either on the biosynthesis of cholesterol (inhibitors of hydroxymethylglutaryl-coenzymeA reductase, statins), or on the capture and elimination of bile cholesterol (sequestrants or resins), or alternatively on lipolysis by a mode of action which remains to be elucidated from the molecular point of view (fibrates). Consequently, all the major categories of drugs which have been used in this indication (sequestrants, fibrates or statins), are designed only for the preventive aspect of the formation of the atheroma plaque and not in fact for the treatment of the atheroma. The current treatment for atheroma, following a coronary accident, are only palliative since they do not act on cholesterol homeostatis and they are surgical acts (coronary by-pass, angioplasty).
The present invention constitutes a new therapeutic approach for the treatment of pathologies linked to dyslipoproteinemias. It proposes an advantageous solution to the disadvantages of the prior art, by demonstrating the possibility of treating pathologies linked to dyslipoproteinemias by gene therapy, by the transfer and expression in vivo of genes capable of regulating the plasma levels of apolipoproteins. The invention thus offers a simple means permitting a specific and effective treatment of these pathologies.
Gene therapy consists in correcting a deficiency or an abnormally (mutation, aberrant expression and the like) by introduction of a genetic information into the cell or affected organ. This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, various techniques exist, of which various techniques of transfection involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255(1980) (10431), and the like. More recently, the use of viruses as vectors for the transfer of genes appeared as a promising alternative to these physical techniques of transfection. In this respect, various viruses were tested for their capacity to infect certain cellular populations, particular the retroviruses (RSV, KMS, MKS, and the like), the HSV viruses, the adeno-associated viruses and the adenoviruses.
The present invention constitutes a new therapeutic approach for the treatment of pathologies linked to dyslipoproteinemias, consisting in transferring and in expressing in vivor genes capable of regulating the plasma levels of apolipoproteins. The present invention also results from the demonstration that adenovirus constitute particularly effective vectors for the transfer and expression of such genes. In particular, the present invention shows that the use of recombinant adenoviruses as vectors makes it possible to obtain sufficiently high levels of expression of these genes to produce the desired therapeutic effect. The present invention thus offers a new approach for the treatment and prevention of cardiovascular and neurological pathologies linked to dyslipoproteinemias.
A first subject of the invention therefore lies in a defective recombinant adenovirus containing at least one inserted gene whose expression makes it possible to regulate the levels of apolipoprotein in vivo.
The subject of the invention is also the use of such a defective recombinant adenovirus for the preparation of a pharmaceutical composition intended for the treatment or for the prevention of pathologies linked to dyslipoproteinemias.
The present invention as more particularly based on the demonstration that the adenovirus type viruses are capable of transferring and expressing genes encoding apolipoproteins in the liver, and of secreting the said apolipoproteins into the circulatory system where they exert their activity. The examples presented later indicate that adenoviruses are capable, according to the mode of administration, of effectively transferring and expressing, for a substantial period and without cytopathological effect, the genes encoding apolipoproteins Al or apoliproprotein AIV.
For the purposes of the present invention, the term xe2x80x9cdefective adenovirusxe2x80x9d designates an adenovirus incapable of autonomously replicating in the target cell. Generally, the genome of the defective adenoviruses used within the framework of the present invention therefore lacks at least the sequences necessary for the replication of the said virus in the infected cell. These regions can be either removed (completely or partially), or rendered non-functional, or substituted by other sequences and especially by the inserted gene. Preferably, the defective virus conserves, nevertheless, the sequences of its genome which are necessary for the encapsulation of the viral particles.
There are various serotypes of adenoviruses, whose structure and properties vary somewhat. However, these viruses are not pathogenic for man, and especially for non-immunosuppressed subjects. Among these serotypes, the use of type 2 or 5 adenovirus (Ad 2 or Ad 5) is preferred within the framework of the present invention. In the case of the Ad 5 adenoviruses, the sequences necessary for the replication are the EIA and EIB regions.
For the purposes of the present invention, the inserted gene capable of regulating the apolipoproteins levels in vivo may be a gene encoding an apolipoproteins or a protein product having an apolipoproteins type activity, or also an antisense gene, whose expression in the target cell makes it possible to control the expression of genes of the transcription of cellular mRNAs encoding apolipoproteins. The term protein product having an apolipoproteins type activity designates any mutant, fragment or peptide having at least one biological property of an apolipoproteins, as well as all natural variants of apolipoproteins.
The inserted gene may be a fragment of cDNA, of genomic DNA, or a hybrid construct consisting for example of a cDNA in which one or more introns would be inserted. It may also be synthetic or semisynthetic sequences.
Among the inserted genes for the purposes of the present invention, there may be mentioned more particularly the genes encoding all or an active part of the apolipoproteins AI, AIV or E.
The apolipoproteins AI is a protein consisting of 243 amino acids, which is synthesized in the form of a prepropeptide of 267 residues, having a molecular mass of 28,000 Daltons. It is synthesized in man specifically in the liver and the intestine and it constitutes the essential protein of the HDL particles (70% of their mass in proteins). It is abundant in plasma (1.0-1.2 g/l). Its best biochemically characterized activity is the activation of lecithin-cholesterol acyltransferase (LCAT), but numerous other activities are attributed to it, such as especially the stimulation of the efflux of cellular cholesterol. The physiological role of apolipoproteins AI appears to be counterbalanced by apolipoproteins AII AI appears to be counterbalanced by apolipoproteins AII since in man, the ratio of the two plasma concentrations (AII/AI) is very closely correlated with coronary risk. Apolipoprotein AI plays a major role in resistance to atherosclerosis, probably linked to the reverse transport of cholesterol, since the sole expression of this apolipoprotein in transgenic mice makes it possible to reduce 40-fold the surface area of the lipid deposits in the aorta compared with control mice (Rubin et al., 1993 Science. In Press). Its gene 1863 bp in length, has been cloned and sequenced (Sharpe et al., Nucleic Acids Res. 12(9) (1984) 3917). Among the protein products with apoliprotein AI type activity, there may be mentioned especially the natural variants described in the prior art (table below).
The apolipoprotein AIV (apoAIV) is a protein consisting of 376 amino acids, which is specially synthesized in the intestine in the form of a precursor of 396 residue. The plasma protein is relatively abundant (0.16 g/l) and has a molecular mass of 46,000 Daltons. It is a major component of the chylomicrons secreted in the lymph, but it has the characteristic feature of being predominant in the form non-associated with lipoproteins in the plasma (R. B. Weinberg, et al., 1983, J. Lipid. Research, 24: 52-59). Moreover, plasma apoAIV is polymorphic, although the nature of this polymorphism is still unknown (G. Utermann et al., 1982, J. Biol. Chem 257: 501-507). The physiological role of apoAIV also remains somewhat unknown. It is known that it can activate, in vitro, lecithin-cholesterol acyltransferase (LCAT) (Steinmetz et al., 1985, J. Biol. Chem., 260: 2258-2264) and that it can, like apolipoprotein AI, interfere with the binding of the HDL particles onto bovine aortic endothelial cells (Savion et al., 1987, Eur. J. Biochem., 257: 4171-4178). These two activities indicate that apoAIV very probably acts as mediator of the reverse transport of cholesterol. The apoAIV gene has been cloned and described in the prior art (see especially WO 92/05253). Among the protein products with apolipoprotein AIV type activity, there may be mentioned especially the fragments and derivatives described in Patent Application FR 92 00806.
Apolipoproteins E comprises 317 residues of which 18 correspond to the signal peptide. There is no peptide. The apoE gene has been cloned and sequenced (about 3600 bp) and encodes an mRNA of 1163 bp, apoE is distributed in the plasma between the VLDL and HDL particles. It represents about 10-20% of the VLDL proteins and 2% of the HDL proteins. The HDL-Es constitute a distinct subclass of HDLs. The plasma concentration of apoE is about 0.05 g/l, apoE is synthesized in the form of sialo-protein which is then desialilated in the plasma. The synthesis of apoE is carried out by the liver and weakly by the intestine. However, contrary to the other apolipoproteins, apoE is also synthesized in numerous other tissues (brain, kidney, adrenal glands, reticuloendothelial cells and the like). apoE recognizes, with a very strong affinity, the LDL receptor (apoB/E receptor) but also another receptor on heptic cells not recognizing apoB (chylomicron/remnant receptor).
A polymorphism was demonstrated on the basis of different electrophoretic mobilities. Six major phenotypes (E2/2, E2/3, E2/4, E3/4, E3/3, E4/4) have thus described. According to the studies carried out on large caucasian populations, the prevalence of the corresponding alleles would be 14-15% for xcex54, 74-78% for xcex53 and 8-12% for xcex52. A difference exists among the Finnish for whom xcex54 is more abundant (23%) and xcex52 is less abundant (4%). The normal allele is xcex53. The xcex52 allele would correspond to the type III dyslipoproteinemia (E2/2 phenotype), a disease which is associated with an increase in cholesterol and triglycerides, xanthomas and with an early atherosclerosis. All association between the xcex54 allele and familial Alzheimer""s disease has been recently reported (Strittmatter et al., P.N.A.S. 99 (1993) 1977). More recently, the destruction of the apoE gene in mice has shown the appearance of a hypersusceptibility to atherosclerosis (E. Rubin et al. Cell 1992).
For the purpose for the invention, the term xe2x80x9cinserted genexe2x80x9d also designates antisense sequences whose expression in the target cell makes it possible to control the expression of genes of the transcription of cellular mRNAs encoding apolipoproteins. Such sequences can for example be transcribed, in the target cell, into RNA complementary to cellular mRNAs and thus block their translation into protein.
Among the antisense sequences which can be used within the framework of the invention, there may be mentioned more particularly any antisense sequence which makes it possible to reduce the levels of production of apolipoprotien AII, as illustrated in the examples.
Generally, the inserted gene also comprises sequences permitting the expression in the infected cell. These may be sequences which are naturally responsible for the expression of the said gene when these sequences are capable of functioning in the infected cell. They may also be sequences of different origin (which are responsible for the expression of other proteins, or even synthetic). In particular, they may be sequences of eukaryotic or viral genes. As example, they may be promoter sequences derived from the genome of the cell which it is desired to infect, or from the genome of a virus, and especially the promoters of the adenovirus genes E1A and MLP, the promoter CMV, LTR-RSV, and the like. In addition, these expression sequences can be modified by addition of activating and regulatory sequences and the like. Moreover, when the inserted gene does not contain expression sequences, it can be inserted into the genome of the defective virus downstream of such a sequence.
Moreover, when the inserted gene encodes an apolipoprotein or a protein product with apolipoprotein type activity, it generally comprises, upstream of the coding sequence, a signal sequence directing the synthesized polypeptide in the secretion pathways of the target cell. This signal sequence may be the natural sequence of the apolipoproteins, but it may also be any other functional signal sequence or an artificial signal sequence.
The defective recombinant adenoviruses according to the invention can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the gene which it is desired to insert. The homologous recombination occurs after cotransfection of the said adenoviruses and plasmid into an appropriate cell line. The cell line used should preferably (i) be transformable by the said elements, and (ii) contain the sequences capable of complementing the defective adenovirus genome part, preferably in integrated form in order to avoid the risks of recombination. As an example of a line, there may be mentioned the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains especially, integrated in its genome, the left-hand part of the genome of an Ad5 adenovirus (12%).
Then, the vectors which have multiplied are recovered and purified according to conventional molecular biology techniques.
The present invention also relates to a pharmaceutical composition containing one or more defective recombinant adenoviruses as described above. Such compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
Preferably, the composition contains vehicles pharmaceutically acceptable for an injectable formulation.
In their use for the treatment of pathologies linked to dyslipoproteinemias, the defective recombinant adenoviruses according to the invention can be administered according to various modes, especially by intravenous injection. Preferably, they are injected at the level of the portal vein.
The doses of virus used for the injection can be adapted according to various parameters, especially according to the mode of administration used, the pathology concerned, the gone to be expressed, or alternatively the duration of treatment desired. Generally, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu/ml and, preferably 106 to 1010 pfu/ml. The term pfu (xe2x80x9cplaque forming unitxe2x80x9d) corresponds to the infectivity of a suspension of virions, and is determined by infection of an appropriate cell culture, and measurement, generally after 48 hours, of the number of placques of infected cells. The techniques for determining the pfu titre of a viral solution are well documented in the literature.
The present invention thus offers a very effective means for the treatment or prevention of pathologies linked to dyslipoproteinemias, in particular in the domain of cardiovascular conditions such as myocardial infarction, angina, sudden death, cardiac decompensation, cerebrovascular accidents, or in the domain of neurological conditions where certain apolipoproteins such as apoE appear to play an important role (diseases of neuronal aging, familial Alzheimer, neuronal regeneration). More generally, this approach offers a very promising therapeutic procedure for each case where a deficiency of a genetic or metabolic nature of a plasma apolipoproteins can be corrected.
In addition, this treatment can be applied both to man and to any animal such as ovines, bovines, domestic animals (dogs, cats and the like), horses, fish, and the like.
The present invention is more completely described with the aid of the following examples which should be considered as illustrative and non-limiting.